Zeng B F, Bounds P L, Steiner R F, Pollack R M
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228-5398.
Biochemistry. 1992 Feb 11;31(5):1521-8. doi: 10.1021/bi00120a032.
The role of Tyr-14 of 3-oxo-delta 5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17 beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site. By analogy, it is argued that the 3-oxo group of steroid substrates for KSI is not protonated during the reaction. In contrast, the fluorescence excitation spectra of 5 bound to KSI show characteristics of an ionized phenol, even at pH values as low as 3.8. It is concluded that the pKa of equilenin is perturbed from its value in solution of 9 to less than or equal to 3.5 at the active site of KSI. Similarly, the pKa of the intermediate dienol in the KSI reaction should be lowered to less than or equal to 4.5 when it is bound to KSI. Thus, the function of Tyr-14 as an electrophilic catalyst is likely the stabilization of the anion of the dienol by hydrogen bonding rather than by proton transfer.
通过分析与3-氧代-δ5-甾体异构酶（KSI）活性位点结合的3-氨基-1,3,5(10)-雌甾三烯-17β-醇（4）和马萘雌酮（5）的光谱，探究了KSI的Tyr-14的作用。与KSI结合的4的紫外光谱与中性溶液中4的紫外光谱相同。这一观察结果表明，Tyr-14在活性位点不会使4的胺基质子化。由此类推，有人认为KSI的甾体底物的3-氧代基团在反应过程中不会被质子化。相反，即使在低至3.8的pH值下，与KSI结合的5的荧光激发光谱也显示出离子化酚的特征。得出的结论是，在KSI的活性位点，马萘雌酮的pKa从其在溶液中的9值扰动至小于或等于3.5。同样，KSI反应中中间二烯醇与KSI结合时，其pKa应降至小于或等于4.5。因此，Tyr-14作为亲电催化剂的功能可能是通过氢键而非质子转移来稳定二烯醇的阴离子。
